In vivo and in vitro aging of common carp Cyprinus carpio sperm after multiple hormonal application and stripping of males.

The present study was designed to evaluate sperm phenotypic variables during in vivo and in vitro storage following multiple sperm stripping in common carp (Cyprinus carpio L.). Each male was injected 3 times with carp pituitary 3 days apart. Sperm was stored in vivo in the body cavity for 0.5 days (Fresh sperm) and 3 days (Old sperm) after hormonal stimulation. Then sperm was collected and diluted with a carp extender at a ratio of 1:1, and stored in vitro on ice for 0, 3, and 6 days. The phenotypic parameters, including the number of total motile spermatozoa, number of fast motile spermatozoa, number of motile spermatozoa, percentage of fast motile spermatozoa, and percentage of spermatozoa motility were the major components of principal component analysis (PCA). In general, Fresh sperm from the first stripping showed slightly better quality than Old sperm from the second and third stripping, especially in the phenotypic parameters of a number of total spermatozoa and a number of total motile spermatozoa (P < 0.05). The highest kinetic and quantitative spermatozoa variables were obtained in Fresh and Old sperm just after sperm collection (0-day storage in vitro), and then they were decreased during the period of in vitro storage up to 6 days (P < 0.05). However, the fertilization, hatching, and malformation rates from Fresh sperm were similar compared with the Old sperm. Sperm could be stripped 0.5 days post hormonal treatment and stored in vitro up to 6 days with good fertilization performance (fertility, hatching, and malformation rates were 92.5%, 91.5%, and 1.3%, respectively). Therefore, our results suggested that multiple hormonal treatments with multiple stripping could be used in artificial reproduction in common carp.

Common carp (Cyprinus carpio) sperm reduction during short-term in vitro storage at 4 °C.

This study was designed to optimize a short-term storage protocol for common carp sperm at 4 °C under aerobic condition. Sperm from individual males were collected directly with or without extenders. The results demonstrated that in general, it was similar effect to collect sperm directly in extenders and keeping sperm for 0.5 h after collection without extenders. Sperm was diluted with eight selected extenders (sperm: extender = 2:1, 1:1 and 1:9) and undiluted sperm was used as a control. Sperm and seminal plasma parameters (sperm motility, velocity, membrane integrity, sperm concentration, osmolality and pH in seminal plasma) were evaluated in sperm stored on ice under aerobic conditions at 0, 2, 4, 6 and 8 days post stripping (DPS) using the computer- assisted sperm analysis system. Results showed that 1:1 and 2:1 dilution maintained higher sperm function and more sperm for a longer period. After 8 DPS, the best sperm quality and quantity was recorded in the common carp seminal plasma supplemented with 50 mM NaCl, Cejko extender (2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, 110 mM NaCl, 40 mM KCl, pH 7.5 and 310 mOsm/kg) supplemented with/without 25 mM KCl/NaCl. The reduction of spermatozoa number with time during short-term storage but varied according to different dilution ratios and extenders. At 8 DPS, sperm count has dropped by 22.9 % in a dilution of 1:1 compared to 50.3 % in sperm without dilution. Extenders with diluted 1:1, especially Cejko solution, largely postponed sperm reduction, 21.3 % compared to 55.5 % for sperm stored without extenders.

Comparison of Quality Changes in Eurasian Perch (Perca fluviatilis L.) Fillets Originated from Two Different Rearing Systems during Frozen and Refrigerated Storage.

The current knowledge on how different Eurasian perch rearing systems impact the final fillet quality is scant. Therefore, two domestic storage conditions were investigated-10 months frozen (-20 °C) and 12 days refrigerated (+4 °C) storage conditions-in order to determine (i) how the choice of rearing system affects fillets quality during different processing conditions and (ii) if oxidative changes and other quality parameters were interactive. For the proposed idea, proteome analysis, oxidative changes, and some quality parameters were considered in this study. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated a higher loss of protein in the frozen fillets from ponds (PF) than the fillets from recirculating aquaculture systems (RAS) (RF). Western blot showed a higher protein carbonyls level in RF compared to PF, which was confirmed by the total protein carbonyls during frozen storage. PF indicated less liquid loss, hardness, and oxidation progress than RF in both storage conditions. The biogenic amines index (BAI) in the fillets from either origin showed acceptable levels during storage at +4 °C. Furthermore, the n-3/n-6 ratio was similar for both fillets. The deterioration of fillets during frozen storage was mainly caused by formation of ice crystals followed by protein oxidation, while protein oxidation was the main concern during refrigerated storage confirmed by principal component analysis (PCA) analysis.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Bleeding of Common Carp (Cyprinus carpio) Improves Sensory Quality of Fillets and Slows Oxidative and Microbiological Changes During Refrigerated Aerobic Storage.

Common carp (Cyprinus carpio) aquaculture is one of the most important and rapidly growing productions around the world. However, for consumers, carp is often not acceptable due to its distinctive colour and odour. In this study, we investigated the effects of bleeding of common carp on fillet quality. The obtained results show that carp bleeding by cutting the gill arches is an effective way of reducing the total haem content, which here decreased from (9.6±1.6) in unbled carp to (2.34±0.8) µmol/kg of haemoglobin in bled carp. Furthermore, fillets from bled carp showed reduced formation of primary and secondary lipid oxidation products and growth of microorganisms during 12 days of refrigerated aerobic storage. On the last day of storage, the amount of lipid hydroperoxides decreased from (88.9±4.2) in unbled to (62.1±2.9) µmol/kg of cumene hydroperoxide in bled carp, TBARS decreased from (4.2±0.5) in unbled to (2.6±0.4) µmol/kg of malondialdehyde in bled carp, mesophilic and psychrotrophic bacteria count decreased from (6.4±0.1) and (6.2±0.3) log CFU/g in unbled to (4.0±0.2) and (4.2±0.2) log CFU/g in bled carp, respectively. These raw bled fillets showed increased lightness L*, and reduced redness a* and yellowness b* compared to unbled fillets. Sensory analysis showed improved colour, odour and overall acceptability of bled raw fillets. Overall, bleeding improves the quality of carp fillets. Thus, inclusion of bleeding into processing of carp fillets has the potential to improve their acceptance by consumers and prolong their shelf-life.

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-µM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

Isolation and transplantation of sturgeon early-stage germ cells.

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 °C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell-specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate-labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P < 0.05). The efficiency of transplantation was 60% (counted 90 dpt).

Morphology and ultrastructure of beluga (Huso huso) spermatozoa and a comparison with related sturgeons.

The ultrastructure and morphology of beluga spermatozoa (Huso huso) (Acipenseridae, Chondrostei) were studied by scanning and transmission electron microscopy. Spermatozoa of 51.27 ± 4.71 µm total length (mean ± SD) were typical of sturgeon and paddlefish spermatozoa. Each consisted of an elongated nucleus (length: 5.84 ± 0.46 µm) with distinct acrosome, a cylindrical midpiece (length: 2.10 ± 0.42 µm), and a single flagellum (length: 42.21 ± 3.82 µm). The acrosome was umbrella shaped (length: 1.12 ± 0.14 µm, width: 0.87 ± 0.10 µm) with seven to nine posterolateral projections (length: 0.49 ± 0.09 µm). Three endonuclear canals, spirally arranged, traversed the nucleus length from the acrosome to the implantation fossa. The flagellum comprised an axoneme with the typical 9+2 organization of microtubules. Two flat fins were present along the flagellum parallel to the plane of the central doublet of microtubules. These fins arose at different positions, 0.57 ± 0.30 and 4.06 ± 1.32 µm from the midpiece and terminated 4.18 ± 1.09 and 4.92 ± 1.34 µm from the distal end of the flagellum. Principal component analysis revealed spermatozoan ultrastructure and morphology were similar among sturgeon species, and, although assigned to genus Huso, beluga may be closely related to the genus Acipenser.